Luminescence is the emission Of photons from electronically excited states. Luminescence can include fluorescence and phosphorescence. In phosphorescence, e.g., from phosphorescent molecule, the photon emission is produced when the excited electrons go from an excited triplet state to an excited singlet state, with the time of return to the ground state from the excited singlet state being relatively long (.mu.sec to sec). In fluorescence, e.g., from a fluorophore, the photon emission is from the excited singlet state to the ground state, and the time required for the return from the excited state to the ground state, is relatively short, with times as short as several nanoseconds and as long as many microseconds. The time required for luminescence photon emission in conjunction with the excited electron's return to the ground state is called the lifetime or decay time.
As used herein, Iuminescencell (or "photoluminescence") refers to one or more of fluorescence, phosphorescence and other photon emissions that are the result of excited elections returning towards the ground state, and "luminescent molecule" refers to a fluorophore, a phosphorescent molecule or a molecule that emits photons when the molecules excited electrons return towards a ground state when excitation is caused by a light source.
In solutions that are at or near room temperature, a variety of molecular events can occur within the nanosecond to microsecond time scale of luminescence which can alter the emission from a luminescent Molecule. Such events can be detected for sensing of analytes by analysis of changes in emission intensity or wavelength, and, in certain cases, by measuring the lifetime of the fluorophore as it changes due to quenching or enhancing effects of an analyte or acceptor molecule that binds an analyte.
In general, sensing of analytes has been conducted using intensity and wavelength shift fluorometry using laser light sources by well-known methods, such as sensing pH using intensity and wavelength shift absorption methods. For example, Moreno et al J. Molec. Struct. 143:553 (1986) describes the fluorescence absorption of cresol red to measure pH; Guthrie et al Talanta 35:157 (1988) uses absorption at two wavelengths via an LED optical pH sensor; Ohkuma et al Proc. Nat'l Acad. Sci. USA 75:3323 (1978) uses fluorescence for pH based on the ratio of intensities at different excitation wavelengths.
Additionally, ph-sensitive fluorophores for intensity or wavelength shift measurements have been described by Saari et al Anal. Chem. 54:823 (1982), which discloses ph-dependent intensity measurements of fluorescein; Wolfbe-Ls et al Fresenius Z. Anal. Chem. 327:347 (1987), which describes pH sensitive probes with varying pH ranges for intensity of: wavelength shift measurements; Offenbacher et al Sensors and Actuators 9:73 (1986), which discloses sensors requiring short wavelength excitation; Wolfbeis et al J. Heterocyclic Chein. 22:1215 (1985), which discloses a variety of pH sensors fol: intensity based measurements; and Wolfbeis et al Fresenius; Z. Anal. Chem. 314:119 (1983), which summarizes fluorescence pH sensors for intensity measurements.
Electroluminescent lamps are potential alternatives to laser light sources, due to the high cost, large size and difficulty in calibration and maintenance of laser light sources. Such alternatives offer the potential for more practical commercial applications in clinical and diagnostic medicine, as well as basic research, due to decreased size, cost, calibration and maintenance.
However, such alternative light sources, when used for intensity- or wavelength shift-based fluorometry, still suffer from the same problems when using intensity- or wavelength shift-based fluorometry with laser light sources, as described herein.
For example, an intensity-based, quenching detecting apparatus is described by Opitz e al. in U.S. Pat. No. 4,889,690. According to this patent, the concentration of oxygen in a sample of water was determined by effectively measuring the oxygen's quenching effect on the intensity of fluorescent radiation emitted by the fluorophores. The fluorescent indicators were excited by continuous light from a luminescent light source, such as a chemoluminescent or an electroluminescent light source, and the intensity of the resulting emitted light radiation (fluorescence) was measured by a photoelectric receiver.
However, the precise and accurate measurement of known intensity- or wavelength shift-based, sensing has suffered from problems such as delayed results, the need for expensive, sophisticated and time consuming procedures that are limited, e.g., by high background noise, low signal-to-noise ratios, turbidity, losses, high light absorbance, photobleaching and fluorophore washout.
Additionally, Lippitsch et al (Anal. Chim. Acta 205:1-6 (1988)) describe the use of the ruthenium complex tris (2,21'-dipyridyl ) ruthenium (II) dichloride hydrate, embedded in silicone, as the active sensing membrane for determination of oxygen quenching of fluorescence lifetimes. The long unquenched sensor fluorescence lifetime of 205 ns in the absence of oxygen allowed the authors to use a relatively inexpensive blue-emitting LED as the excitation light source. However, since the optical output power of the commercially available blue-emitting silicon carbide LEDs is rather low, extremely sensitive (and expensive) light detectors, such as photomultipliers, are required to measure the emitted fluorescence radiation.
All of the above problems have the potential to be eliminated by the use of apparatus and methods wherein changes in decay time, phase angle and/or modulation of emitted luminescence are detected as parameters that correspond to changes in luminescence lifetimes, rather than to changes in luminescence intensity.